Gene disruption in Schizosaccharomyces pombe using a temperature-sensitiveUra4p

We have generated a temperature-sensitive form of the Ura4p protein from th e fission yeast Schizosaccharomyces pombe. A single T-to-C mutation at nucl eotide 782 (relative to the initiator ATG codon of ura4) changes the leucin e residue at position 261 in Ura4p to a proline. The mutant Ura4p(ts) suppo rts growth at 30 degrees C but is unable to allow growth at 37 degrees C in the absence of uracil when a single copy of the gene is integrated into th e host chromosome. Using the ura4(ts) cassette for gene replacements simpli fies the identification of transformants in which the disruption construct has undergone homologous integration into the host chromosome, as these ind ividuals contain a single copy of the ura4(ts) gene and fail to grow when r eplicated to 37 degrees C in the absence of uracil. Copyright (C) 1999 John Wiley & Sons, Ltd.